MODULATION OF CALCIUM CONTROL OF CARDIAC MYOFIBRILS

心脏肌原纤维钙控制的调节

• 批准号: 3336761
• 负责人: R John Solaro
• 金额: $ 16.64万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3336761
• 关键词: acid base balance; acidity /alkalinity; actins; adenosine diphosphate; adenosinetriphosphatase; affinity chromatography; calcium binding protein; calcium metabolism; calcium transporting ATPase; cations; ethylene glycols; homeostasis; hydrogen; hypoxia; ion transport; ischemia; laboratory rat; lactates; magnesium; membrane permeability; muscle relaxants; muscle stimulant; muscle tension; myocardium; myofibrils; myosins; nucleotides; phosphorylation; stretch reflex; troponin

The general goals of the proposed research has been to understand the process by which Ca2+ activates the mechanical and biochemical activity of cardiac myofilaments, and to known whether, how, and when this process is modified physiologically by intrinsic mechanisms and by mechanisms extrinsic to the myofilaments involving protein phosphorylation. Experiments relating to intrinsic regulation are aimed at determination of whether Ca2+ binding to regulatory sites in the myofilament lattice are influenced by the sarcomere length and/or by force-generating cross-bridge complexes with the thin filament. Experiments relating to extrinsic regulation focus on phosphorylation of troponin I (TnI), the P-light chain of myosin, and C-protein. The aims are to determine the effect of TnI phosphorylation on the binding of cross-bridges to the thin filament, and also to determine whether there are isoforms of TnI and how variations in the population of the TnI isoforms affect myofilament activity and regulation. In the case of studies on C-protein and the P-light chain of myosin, the aims are to determine whether these proteins affect myofilament activation by Ca2+ and whether their activity is influenced by the level of protein phosphorylation. The approach to these aims involves measurement of levels of covalent phosphorylation of myofilament proteins in hearts freeze-clamped during the response to inotropic perturbations. The approach also involves preparation of each of the main myofilament proteins, analysis of the isoforms of TnI and myosin heavy chain, reconstitution with variations in the levels of covalent phosphorylation and measurement of cross-bridge binding to actin and actomyosin ATPase activity. Mechanical studies using chemically skinned fibers include measurements of tension, stiffness, Vmax, and kinetics of the rise and fall of force during "jumps" and "dives" in the free Ca2+ surrounding the myofilaments. Methods also include newly developed procedures for making multiple measurements of Ca2+ binding to chemically skinned preparations of heart muscle, characterized for TnC content, sarcomere length and extent of actin-cross-bridge interaction. The proposed experiments bear directly on current perceptions of the regulation of cardiac output by the Starling mechanism and by the autonomic nervous system, and whether extrinsic and intrinsic regulation of the Ca2+ activation of the myofilaments might represent a locus of lesions in heart failure.

拟议研究的一般目标是了解 CA2+激活的过程 心脏肌丝，并知道该过程是否，如何以及何时 通过内在机制和机制进行生理修饰 涉及蛋白质磷酸化的肌膜外丝。 与内在调节有关的实验旨在确定 Ca2+与肌丝晶格中的调节位点结合是否 受肌节长度和/或受力生成的杂交桥的影响 与细丝的复合物。 与外部有关的实验 调节肌钙蛋白I（TNI）的磷酸化（P-Light链）的磷酸化 肌球蛋白和C蛋白。 目的是确定TNI的影响 跨桥与细丝的结合，磷酸化， 还确定是否有TNI同工型以及如何变化 TNI同工型的种群影响肌丝活动和 规定。 在研究C蛋白和P-Light链的情况下 肌球蛋白，目的是确定这些蛋白是否影响肌膜 Ca2+的激活以及它们的活性是否受到水平的影响 蛋白质磷酸化。 这些目标的方法涉及测量 心脏中肌丝蛋白的共价磷酸化水平 在对肌力扰动的反应过程中，冻结夹。 这 方法还涉及准备每个主要肌丝 蛋白质，分析TNI和肌球蛋白重链的同工型， 重新构造，共价磷酸化水平的变化 并测量与肌动蛋白和肌动蛋白ATPase结合的桥桥结合 活动。 使用化学皮肤纤维的机械研究包括 张力，刚度，VMAX和动力学的升高和下降的测量值 在周围的自由Ca2+中的“跳跃”和“潜水”期间 肌膜。 方法还包括新开发的程序 Ca2+与化学皮肤制剂的多次测量 心肌，特征在于TNC含量，肌节的长度和范围 肌动蛋白 - 交叉桥的相互作用。 提出的实验直接在 当前对Starling对心输出量调节的看法 机制和自主神经系统，以及外部和外在的 肌膜的Ca2+激活的固有调节可能 代表心力衰竭病变的基因座。